( attempts resulted in libraries that are...)Constructing Directional cDNA Libraries from Limited Amounts of RNA

Stratagenes new PCR Library Construction Kit can use as little as 50 ng of total RNA as starting material to construct directional libraries in plasmid vectors. The method featured in this kit combines PCR for amplifying cDNA synthesized from small amounts of RNA with LIC for high-efficiency, directional cloning.

The kits double-stranded cDNAs are synthesized, then ligated to an adaptor that contains a sequence for LIC. The adaptor-ligated cDNA is amplified by PCR using primers with specifically designed LIC ends.^1-3 The PCR products are ready for purification and directional cloning into a vector with compatible LIC overhangs. The kit offers numerous advantages over other cloning methods: Pfu DNA polymerase, which ensures the highest possible fidelity of the amplification process, can be used to amplify cDNA inserts. Because cloning of PCR products is directional, libraries generated using this method can be used for high-throughput 3 EST sequencing and for functional screening. Additionally, this method does not depend on the use of T4 DNA ligase to covalently link the vector and insert; consequently, the background of self-ligated vector is reduced. Since PCR products are cloned without endonuclease reactions, cleavage of inserts at internal restriction sites is avoided.

On another exciting front, the kit can be used to make cDNA libraries from tissue samples isolated by laser-capture microdissection (LCM).⁴ This technique, developed at the National Institutes of Health, captures pure populations of targeted cells from microscopic samples of tissue samples for transfer to a polymer film. Specific cells are isolated within their own milieu, thus securing the complex biochemical and physical effects exerted by the surrounding environmen
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